Antibodies to human neutrophil-specific antigens (HNA) were shown to cause clinical complications such as pulmonary transfusion reactions and in some cases transfusion related acute lung injury (TRALI) (Popovsky et al. Am. Rev. Resp. Dis. 128(1): 185-9, 1983) or causing neonatal alloimmune neutropenia (NAIN) (Bux, et al. Transfus. Med. 2(2): 143-9, 1992). Therefore, detection of HNA specific antibodies has important clinical applications.
TRALI is a life-threatening transfusion complication and is one of the most frequent causes of transfusion-related death in the United States. TRALI is the second most frequent transfusion-related cause of death in Europe after administration of ABO-incompatible stored blood (Holness et al. Transfus Med. Rev. 18: 184-188, 2004). The risk of developing TRALI as a complication of blood transfusion is at least 2000-times higher than contracting an HIV- or hepatitis C infection.
TRALI is defined as a clinical entity consisting of sudden acute shortness of breath within six hours after blood transfusion, connected with bilateral lung infiltrations (lung edema) with no indication of cardiac insufficiency or volume overload (European Haemovigilance Network (EHN). Definitions of Adverse Transfusion Events available on the EHN web site).
TRALI syndrome is difficult to diagnose, because initially it often does not differ from a transfusion-independent lung insufficiency (ALI) or its maximum variant ARDS (“acquired respiratory distress syndrome”) (Popovsky & Moore, Transfusion 25: 573-577, 1985). Symptoms of TRALI include hypoxemia, tachycardia, hypotension, cyanosis and fever. Often, TRALI is not recognized or misdiagnosed in the clinic because the symptoms are often attributed to other causes, such as fluid overload. TRALI has been associated with the transfusions of all plasma containing blood components, including whole blood, red blood concentrates, fresh frozen plasma, whole blood derived from platelets, pooled platelets, intravenous gamma-globulin, cryoprecipitate, stem cells and granulocytes. TRALI is an injury to the pulmonary microvascular; and therefore, treatment focuses on respiratory support and saline infusion.
TRALI is an immune-related disorder that is primarily associated with antibodies specific for HNA, granulocyte- and human leukocyte antigens (HLA) Class I. Other factors that have induced TRALI in transfusion recipients include biologically active lipids and HLA Class II antibodies. In most cases, antibodies of the donor (in the donor plasma) are transferred with the stored blood and then react with the leucocytes (granulocytes) of the recipient. The binding of the antibodies to the granulocytes leads to their activation and partially to aggregation. Through the subsequent release of the microbicidal arsenal from the granulocytes, the capillary endothelium is damaged which results in lung edema. The immune reaction induces complement-activated granulocytes to release oxygen radicals and proteases that damage the endothelium resulting in the extravasation of protein-rich fluid into the pulmonary alveoli and interstitium. In addition, antibodies within stored blood will bind to and activate granulocytes of the recipient resulting in the expression of adhesion molecules (Uchiyama et al. Transfus. Med. Rev. 8: 84-95, 1994), transmigration of granulocytes into the interstitial space between alveolar and vessel endothelium of the lung, and the release of cytokines, proteases and oxygen radicals (Snyder, Immol Invest. 24: 333-9, 1994). These cellular effects cause damage to the capillary walls with subsequent hyperpermeability. A lung edema develops and 10% of the affected patients die from this complication.
In TRALI, recipient antibodies rarely react with the granulocytes of the donor (Bux et al., Br. J. Heamatol. 93: 707-713, 1996). However, there have also been cases of TRALI that were induced by antibodies in the transfusion recipient. In very rare cases, anti-IgA-antibodies can also induce TRALI (Saigo et al., J. Int. Med. Res. 27: 96-100, 1999).
Blood donations of multiparous women carry particular risk, because an antibody formation against granulocyte- or HLA-antigen of the child can occur during the pregnancies. Likewise, a patient may be immunizing due to an earlier transfusion (Voss et al., Anaesthesist 50: 930-932, 2001). Donor plasma that will trigger TRALI cannot be detected clinically. Currently produced erythrocyte concentrates contain very little plasma and only a few granulocytes, therefore TRALI is most likely to occur after administration of fresh plasma and platelet concentrates.
In addition to HLA antibodies, antibodies against three different antigen systems on granulocytes are thought to be responsible for inducing TRALI (Leger et al. Anesthesiology 91: 1529-1532, 1999; Davoren et al, Transfusion 43: 641-645, 2003; Kopko et al. JAMA 287: 1968-1971, 2000; Reil et al. Vox Sanguinis (printing, already accessible online), 2008. Two of the antigen systems (HNA-1 and HNA-2) are known with regard to their structure and localization. The antigen HNA-2 was characterized by Prof. Dr. Bux and applied for as a patent (DE 100 28 725 A1). The third antigen system, HNA-3 (consisting of the antithetic antigens HNA-3a and HNA-3b), has not been characterized. The antigen HNA-3a occurs in approximately 95% of the population (Davoren et al, Transfusion 43: 641-645, 2003) and is involved particularly frequently in severe courses of TRALI (Reil et al., Vox Sanguinis (printing, already accessible online), 2008).
According to the current report by SHOT (Serious Hazards of Transfusion), the British notification and evaluation centre for side-effects in blood transfusions, TRALI is the most frequent cause of a serious side-effect due to transfusion. The report shows a mortality of 9% for the period 1996-2003 (SHOT), Additional cumulative data 1996-2003 is available at the Serious Hazards of Transfusion (SHOT) web site). Since 2001, United States Food and Drug Administration likewise reported TRALI as the main cause of transfusion-associated complications (Goldman et al., Transfus. Med. Rev. 19: 2-31, 2005; Boshkov, Vox Sang. 83: 299-303, 2002).
Currently, most blood and tissue donors have not been HNA typed. The specialized nature of neutrophil immunobiology, the scarcity of HNA typing sera and the need to test fresh neutrophils places restraints on typing HNA compatible blood components. A high percentage of cases of TRALI are caused by blood donated by females, particularly multiparous females, and from the transfusion of fresh frozen plasma. Proposed current solutions for reducing the incidence of TRALI include the exclusion of all females as donors, to exclude multiparous (three or more pregnancies) females as donors, and reducing the transfusion of fresh, frozen plasma
Currently, the detection of granulocyte-specific antibodies is laborious; and detection of HLA antibodies in the serum of the blood donor is not sufficient. The most reliable determination of a TRALI risk currently consists in a cross-matching between donor serum and patient leucocytes. This test can only be carried out in specialized laboratories (Voss, Anaesthesist 50: 930-932, 2001) which are not suitable for donor screening. Other strategies are currently directed to a more restrictive donor management (Mair et al., Crit. Care Med. 34: 137-143, 2006) (as described above). This is not acceptable because the exclusion of women from blood donation after a pregnancy leads to a serious reduction in the amount of stored blood.
The exclusion of female donors was investigated systematically in Canada. Through the exclusion of multiparous female donors, 12% of all blood donations would be omitted from the Canadian Blood Service (Goldman et al Transfus. Med. Rev. 19: 2-31, 2005). According to some studies, implementing such a strategy would exclude every third potential female donor. (Densmore et al., Transfusion 39: 103-106, 1999). An alternative strategy would be the testing of all stored blood for granulocyte-specific antibodies. Currently, this technically cannot be carried out. Other strategies for processing the blood components are proposed, but these strategies would involve new risks such as bacterial contaminations and due to their time requirement are only suitable for planned transfusions, and not for those in emergencies (Mair et al., Crit. Care Med. 34: 137-143, 2006). Furthermore, evidence is lacking as to whether such strategies can actually reduce the incidence of TRALI.
Human neutrophil antigens are also known as neutrophil-specific antigens or HNA. Currently there are 5 HNA antigen systems: HNA-1, HNA-2, HNA-3, HNA-4 and HNA-5. Alleles for HNA-1, 2, 4 and 5 were identified and the corresponding glycoproteins were characterized; however, the allele for HNA-3 remains unknown (reviewed by Stroncek, ASHI Quarterly 2004). There are three HNA-1 antigens (HNA-1a, HNA-1b and HNA-1c) that are expressed solely on neutrophils and are located on low affinity Fc-γ receptor IIIb. The HNA-2 system has one well established antigen (HNA-2a). HNA-2 is only expressed on neutrophils and neutrophil precursors and is located on the glycoprotein CD177 (NB1 gp). HNA-4 and HNA-5 are located on the β2 integrin. HNA-4 is expressed on granulocytes, monocytes and lymphocytes. (See Stroncek, ASHI Quarterly 2004)
The HNA-3 system has one known antigen, HNA-3a, which is also known as 5b. HNA-3 is expressed on neutrophils, lymphocytes, platelets, endothelial cells, kidney, spleen and placenta cells, and is known to be located on a 70 to 95 kDa neutrophil glycoprotein. (See Stroncek, ASHI Quarterly 2004). The gene for HNA-3a has not been cloned and the nature and function of glycoprotein was previously unknown. Therefore, current detection of HNA-3 antibody is only based on non-specific assays, such as agglutination tests (Lalezari & Bernard, Transfusion 5: 135-42, 1965) or GIFT-FC assay (Davoren et al., Transfusion 43(5): 641-5, 2003).
The presumed allele of HNA-3, also known as 5b, has a gene frequency about 0.82 (Van Leeuwen et al. Vox Sang 9: 431-46, 1964). It was also reported to have a 0.66 gene frequency (Lalezari & Bernard, Transfusion 5: 135-42, 1965). The protein of 5b was reported to have a molecular weight of 70 to 95 kD (De Haas et al, Transfusion 40(2): 222-7, 2000), yet the 5b gene has not been cloned and the nature and function of the protein remain unknown.
Of interest to the present invention is CTL2 is a 706 amino acid membrane-spanning protein (about 80.152 kD) that comprises 10 helical transmembrane domains. This protein is also known as which is also known as SLC44A2, DKFZp666A071 2, FLJ44586 2 and PP1292 and is known to be involved in choline transport within the inner ear and is expressed on inner ear supporting cells. The gene encoding CTL2 is located on chromosome 19p13. In addition, the antigen, Inner Ear Supporting Cell Antigen (IESCA) is known to be a CTL2 protein which is reactive with an autoantibody associated with autoimmune sensorineural hearing loss (AISNHL).
Currently, the methods of screening and typing transplant tissue or transfusions for HNA antibodies that induce TRALI are inadequate and problematic. In addition, excluding a large portion of the human population from donating blood and tissue is an extreme solution. Therefore, a strong need exists for the development of methods of screening for HNA antigens.
It was therefore an object of the present invention to clarify the protein- or DNA sequences of the human neutrophil antigen-3a or -3b (HNA-3a, HNA-3b) involved in TRALI and to provide the corresponding sequences.